Accelerated development of TEM-29 extended-spectrum beta-lactamase from TEM-1 in hypermutable, mutS, Escherichia coli

Abstract number: 1134_01_68

Ellington M.J., Livermore D.M., Pitt T.L., Hall L.M.C., Woodford N.

Objectives:  

Lesions in the DNA-repair pathway gene mutS can result in a hypermutable phenotype in E. coli, and this may expedite the diversification of beta-lactamases. To gain significant activity against ceftazidime (CTZ), at least one amino acid substitution must occur in the TEM-1 beta-lactamase. In order to examine the effect of hypermutability on extended-spectrum beta-lactamase (ESBL) development from TEM-1, we compared the rate of CTZ resistance development from blaTEM-1 in wild-type and mutS-negative E. coli.

Methods:  

The XmnI and BsrFI fragment of pBR322 replaced the corresponding fragment of pUC19, creating a pUC19-based vector encoding classical TEM-1; designated pTEM-1. CTZ MICs for isogenic wild-type and mutS-negative E. coli harbouring pTEM-1 were determined by Etest. Selection of mutants resistant to 4 × MIC CTZ was performed in triplicate experiments from 3 independent cultures on agar, and the frequency of CTZ resistance was calculated. MICs for randomly-selected colonies were determined. Mutations in blaTEM-1 were identified by sequencing.

Results:  

MICs of CTZ for the wild-type and mutS-negative parent strains harbouring pTEM-1 were 0.25 mg/ml. The emergence rate of CTZ-resistant mutants was >300-fold higher for the mutS-negative strain (1.58 × 10^­6, SE 8.55 × 10^­7) than for the wild-type (4.14 × 10^­9, SE 8.43 × 10^­10). Thirteen of 19 mutants from the mutS-negative host were CTZ resistant at the British Society for Antimicrobial Chemotherapy breakpoint of >2 mg/ml (range 4–16 mg/ml). DNA sequencing showed an Arg164His substitution in all of 4 sequenced mutants. This mutation changes TEM-1 into the ESBL TEM-29. All of 20 mutants selected from the wild-type host were susceptible to <2 mg/ml CTZ, and, none of these had mutations in the blaTEM-1 gene.

Conclusions:  

Resistance-conferring point mutations occurred readily within blaTEM-1 in a mutS background but did not occur at a detectable frequency in a wild-type background. Whilst the role of hypermutators in ESBL evolution is uncertain, these experiments indicate its potential.